The mitochondrial permeability transition (MPT) refers to a transition in the permeability of the inner mitochondrial membrane to solutes with molecular mass below approximately 1500 Da. The MPT is thought to be mediated by the reversible opening of a voltage and Ca2+-dependent, high conductance, protein channel in the inner mitochondrial membrane, the MPT pore (MPTP). The consequences of MPTP opening are twofold: firstly, there is uncoupling of oxidative phosphorylation and as a consequence the F1F0-ATPase reverses to try and maintain mitochondrial membrane potential (ΔΨm) which results in a decline in cellular ATP levels and a loss of metabolic homeostasis. Secondly, the MPTP allows solutes to freely enter the mitochondrial matrix which results in swelling and eventual rupture of the outer mitochondrial membrane with subsequent release of stored calcium and proapoptotic factors. The release of stored calcium can cause calcium overload, production of reactive oxygen species (ROS) and MPT in neighbouring mitochondria resulting in a “chain reaction” throughout the cell. Depending on the energy status of the cell, apoptosis or necrosis then occurs leading to irreversible tissue and organ damage (Grimm S., Brdiczka D. The permeability transition pore in cell death Apoptosis, 2007, 12, 841-847).
The precise molecular composition of the MPTP is still not known. Cyclophilin D has been shown both pharmacologically (using an inhibitor, Cyclosporin A) and genetically to be a major regulator of the MPTP. Many studies demonstrating a role for the MPTP in disease have been conducted using cyclophilin D null mice (Ppif−/−) or Cyclosporin A. The MPTP can be regulated by several factors including, high [Ca2+], oxidative damage (by ROS), chemical cross-linking agents, stress signalling and the PI3-kinase signalling pathway, conditions which are often present in the cells from diseased tissues (Rasola, A., Bernardi, P., The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis. Apoptosis, 2007, 12, 815-833).
The role of mitochondria-mediated apoptosis and necrosis in the aetiology of many diseases is well established and the increased rate of apoptosis/necrosis typical of pathological stress conditions such as those observed during myocardial infarction, renal ischemia, or neurodegenerative diseases correlates with the MPT and loss of mitochondrial integrity.
Hence the MPTP has been implicated in the aetiology and progression of several diseases including:                Acute Myocardial Infarction (Lethal Reperfusion Injury);        Stroke and Neurological Diseases;        Inherited dystrophies;        Hepatitis;        Diabetes and Diabetic retinopathy.Acute Myocardial Infarction (Lethal Reperfusion Injury)        
In ischemic heart disease, sequential ischemia-reperfusion events occur resulting in myocardium cell death by necrosis and/or apoptosis. Lethal reperfusion injury, (cardiomyocyte death as a direct result of tissue reperfusion) is thought to account for up to 50% of the final myocardial infarct size and has been shown to be dependent on the RISK (Reperfusion Injury Salvage Kinase) pathway and MPTP opening (Yellon, D. M., Hausenloy, D. J., Myocardial Reperfusion Injury. N. Engl. J. Med., 2008, 357, 1121-1135.). During ischemia there is depletion of ATP, a drop in cellular pH and intracellular loading of Ca2+. At reperfusion the increased intracellular [Ca2+] leads to Ca2+ overload in the mitochondria which together with a large burst of ROS and a return to physiological pH causes, paradoxically, opening of the MPT and cardiomyocyte cell death via necrosis and/or apoptosis. Thus, inhibition of the MPTP would be expected attenuate cardiomyocyte death and reduce infarct size after ischemia/reperfusion injury. Indeed, Debio-025, a cyclosporin A analogue endowed with MPT inhibitory activity, reduced infarct size and improved functional recovery and mortality in a mouse model of myocardial infarction and reperfusion injury (Gomez, L., Thibault. H., Gharib, A., Dumont, J-M., Vuagniaux, G., Scalfaro, P., Derumeaux, G., Ovize, M. Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice. Am. J. Physiol. Heart Circ. Physiol., 2007, 293, 1654-1661).
Stroke and Neurological Diseases.
Cerebral ischemia followed by reperfusion activates several pathways including one that causes the release of large quantities of the excitatory amino acid glutamate into the synapses. Activation of N-methyl-D-aspartate receptors causes an increase in calcium uptake and ROS production leading to opening of the MPTP and mitochondrial dysfunction. Hence MPTP opening has been implicated in the neuronal cell death and clinical symptoms associated with stroke. Further, NIM811, a cyclosporin A analogue MPT inhibitor, afforded 40% protection in a rat model of transient focal cerebral ischemia characterized by a reduction in cytochrome C release and ROS production (Korde, A. S., Pettigrew, L. C., Craddock, S. D., Pocernich, C. B., Waldmeier, P. C., Margolis., W. F. Protective effects of NIM811 in transient focal cerebral ischemia suggests involvement of the mitochondrial permeability transition. J. Neurotrauma, 2007, 24 (5), 895-908).
Severe insulin-induced hypoglycaemia causes neuronal damage to selective regions of the brain including the outer layers of the cortex and the dentate gyrus. In a rat model of insulin-induced hypoglycaemic coma Cyclosporin A, an immunosuppressant drug endowed with MPTP inhibitory activity, but not FK506 (an immunosuppressant similar to CSA but devoid of MPTP activity) showed a robust reduction in ultra-structural brain damage when administered 30 minutes prior to hypoglycaemic insult (Friberg, H., Ferrand-Drake, M., Bengtsson, F., Halestrap, A. P., Wieloch, T. Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycaemic damage and implicates the mitochondrial permeability transition in cell death. J. Neurosci., 1998, 18, 5151.5159).
Mitochondrial dysfunction, aberrant Ca2+ signalling and oxidative stress are characteristic of Amyotrophic Lateral Sclerosis, Alzheimer's, Parkinson's and Huntington's diseases and MPTP opening has been causally linked to all four diseases by the selective use of Cyclosporin A (Norenberg, M. D., Rama Rao, K. V. The mitochondrial permeability transition in neurologic disease. Neurochem. Int., 2007, 50, 983-997).
MPTP and mitochondrial swelling has also been implicated in brain damage resulting from hyperglycaemic insult, experimental trauma and epilepsy (Li, P. A., Uchino, H., Elmer, E., Siesjö, B. K. Amelioration by cyclosporin A of brain damage following 5 or 10 min of ischemia in rats subjected to preischemic hyperglycaemia. Brain Res., 1997, 753, 133-140; Scheff, S. W., Sullivan P. G. Cyclosporin A significantly ameliorates cortical damage following experimental traumatic brain injury in rodents. J. Neurotrauma, 1999, 16, 783-792; Kudin, A. P., Debska-Vielhaber, G., Vielhaber, S., Elger, C. E., Kunz, W. S. The mechanism of neuroprotection by topiramate in an animal model of epilepsy. Epilepsia, 2004, 45, 1478-1487).
Inherited Dystrophies
Many of the pathological features leading to MPTP opening (Ca2+ overload and ROS accumulation) are present in muscular dystrophies and mitochondria isolated from skeletal muscle from Scgd−/− mice (model of severe dystrophy) are swollen consistent with opening of the MPTP. Scgd−/− Ppif−/− mice (devoid of cyclophilin D) did not have swollen mitochondria and did not exhibit severe muscle degeneration at 8 weeks of age. Further, treatment of mdx (model of Duchene muscular dystrophy) and Scgd−/− mice with Debio-025 reduced mitochondrial swelling and necrotic disease (Millay, D. P., Sargent, M. A., Osinska, H., Baines, C. P., Barton, E. R., Vuagniaux, G., Sweeney, H. L. Robbins, J., Molkentin, J. D. genetic and pharmacologic inhibition of mitochondrial-dependent necrosis attenuates muscular dystrophy. Nature Medicine, 2008, 14, 442-447).
Ullrich congenital muscular dystrophy and Bethlem myopathy are two inherited dystrophies characterized by mutations in the collagen VI gene. Mice models of the diseases have highlighted a latent mitochondrial dysfunction characterized by increased susceptibility to MPTP opening and myofibre degeneration. Myoblasts from patients with Ullrich congenital muscular dystrophy showed mitochondrial dysfunction and precocious opening of the MPTP leading to increased apoptosis. Patients with Collagen VI myopathies treated with the MPTP inhibitor Cyclosporin A for one month showed amelioration of mitochondrial function and had signs of muscle regeneration (Merlini, L., Angelin, A., Tiepolo, T., Braghetta, P., Sabatelli, P., Zamparelli, A., Ferlini, A., Maraldi, M., Bonaldo, P., Bernardi, P. Cyclosporin A corrects mitochondrial dysfunction and muscle apoptosis in patients with collagen VI myopathies. P.N.A.S., 2008, 105 (13), 5225-5229).
Hepatitis
Liver can be damaged by different agents such as chemical poisons, inflammatory factors or viruses. In all cases, hepatocytes undergo massive apoptosis that is driven by the MPTP. Furthermore, it has been reported that inhibiting MPTP opening by treatment with cyclosporine A strongly reduces liver damage in a rat model of TNF-α-dependent acute inflammatory hepatitis (Soriano, M. E., Nicolosi, L. Desensitization of the permeability transition pore by cyclosporine A prevents activation of the mitochondrial apoptotic pathway and liver damage by tumour necrosis factor-alpha. J. Biol. Chem., 2004, 279, 36803-36808).
Diabetes
Diabetes induces damage and cell death by several mechanisms. Diabetic retinopathy (DR) is one of the peripheral micro-vascular complications strongly enhancing the morbidity of diabetic vascular diseases. DR begins with an early pre-proliferative stage (background retinopathy) characterized by loss of capillary pericytes, progressive capillary closure, micro-aneurysms and retinal oedema. The subsequent retinal ischemia (or hypoxia) due to vessel occlusion, triggers abnormal retinal vessel growth. Neo-vessels extend along the inner surface of the retina and/or into the vitreous cavity and can lead to retinal detachment and haemorrhage. This stage is known as proliferative diabetic retinopathy (PDR). Hyperglycaemic stress is considered a key factor in PDR since it induces increased production of vascular endothelium growth factor (VEGF) by retinal cells leading to neovascularization and causes cellular oxidative damage having repercussions on the mitochondria. ROS, formed in higher amounts during diabetes, could trigger most of the pathologic intracellular pathways involved in PDR and it has been demonstrated that ROS are produced in retina during reperfusion following diabetes-induced ischemia. In addition, oxidative stress was also correlated with incidence and progression of retinopathy of prematurity (ROP). The immature retina contains relatively low levels of antioxidants such as heme-oxygenase-1 and catalase. During hyper-oxygenation ROS are produced and, among other things, favour the generation of biologically active isoprostanes concurring to ischemia and, therefore, to the pathogenesis of ROP. The MPTP is triggered by ROS and its opening can lead to the further production of ROS and so mitochondrial dysfunction could be central to diabetic complications.
To date, inhibition of the MPTP has been mainly restricted to pharmacological modulation of the cyclophilin-D component of the MPTP using the potent immunosuppressant drug cyclosporine-A or its analogues NIM811 and Debio-025 (that are devoid of immunosuppressant activity). These are large, complex molecules based on the peptidic structure of cyclosporine-A. In addition, the inhibitory efficacy of these molecules is restricted by the limits of the regulatory role of cyclophilin D in MPTP function and its level of expression.
Other known compounds, which often show a variety of biological activities and pharmacological profiles, have been reported to have some additional non-specific interactions also with the MPTP. As an example, the agent N-[(3,5-di-tert-butyl-4-hydroxy-1-thiophenyl)]-3-propyl-N′-(2,3,4-trimethoxybenzyl)piperazine (S-15176) has been shown to interact with several targets on rat liver mitochondrial membranes and displays some anti-ischemic properties when dosed in vitro at low concentration. Conversely, at higher doses it induces depolarization of the mitochondrial membrane and respiration uncoupling, which are typically associated to severe cytotoxicity. (Morin D. et al. Effect of the mitochondrial transition pore inhibitor, S-15176, on rat liver mitochondria: ATP synthase modulation and mitochondrial uncoupling induction. Biochemical Pharmacology, Pergamon, Oxford, GB, 72, 7, 911-91).
Hence there is a need to identify more potent and effective small molecule inhibitors of the MPTP having a different and more efficacious target within the MPTP which are useful in the prevention or therapy of diseases and conditions associated with the activity of the MPTP.
The acrylamido compounds of the present invention are small molecules endowed with potent MPT inhibitory activity, which are useful in the treatment of a variety of diseases such as those resulting from ischemia/reperfusion damage or oxidative damage, age-related diseases, degenerative and neurodegenerative diseases.
Certain acrylamido compounds are known in the art as therapeutic agents.
As an example, aryl and heteroaryl propene amides have been disclosed as antiproliferative, radioprotective and cytoprotective agents in the patent application WO 04/037751. Acrylamido compounds ligand of vanilloid receptor have been disclosed in the patent application WO 03/049702 as analgesics for the treatment of pain of various genesis and etiology. N-heterocyclyl amide compounds serotoninergic antagonists have been disclosed in the patent application WO 01/068585 for the treatment or prevention of central nervous system disorders. In addition, certain caffeic acid anilides are also known in the art as anti-platelet aggregation and anti-oxidative agents, as reported in Bioorganic & Medicinal Chemistry 2005, 13(5), 1791-7.